From the Department of Microbiology, Immunology, and Molecular Genetics, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, University of California, Los Angeles, California 90095.
J Biol Chem. 2014 Feb 21;289(8):4723-34. doi: 10.1074/jbc.M113.482067. Epub 2014 Jan 8.
mTORC1 plays critical roles in the regulation of protein synthesis, growth, and proliferation in response to nutrients, growth factors, and energy conditions. One of the substrates of mTORC1 is 4E-BP1, whose phosphorylation by mTORC1 reverses its inhibitory action on eIF4E, resulting in the promotion of protein synthesis. Raptor in mTOR complex 1 is believed to recruit 4E-BP1, facilitating phosphorylation of 4E-BP1 by the kinase mTOR. We applied chemical cross-linking coupled with mass spectrometry analysis to gain insight into interactions between mTORC1 and 4E-BP1. Using the cross-linking reagent bis[sulfosuccinimidyl] suberate, we showed that Raptor can be cross-linked with 4E-BP1. Mass spectrometric analysis of cross-linked Raptor-4E-BP1 led to the identification of several cross-linked peptide pairs. Compilation of these peptides revealed that the most N-terminal Raptor N-terminal conserved domain (in particular residues from 89 to 180) of Raptor is the major site of interaction with 4E-BP1. On 4E-BP1, we found that cross-links with Raptor were clustered in the central region (amino acid residues 56-72) we call RCR (Raptor cross-linking region). Intramolecular cross-links of Raptor suggest the presence of two structured regions of Raptor: one in the N-terminal region and the other in the C-terminal region. In support of the idea that the Raptor N-terminal conserved domain and the 4E-BP1 central region are closely located, we found that peptides that encompass the RCR of 4E-BP1 inhibit cross-linking and interaction of 4E-BP1 with Raptor. Furthermore, mutations of residues in the RCR decrease the ability of 4E-BP1 to serve as a substrate for mTORC1 in vitro and in vivo.
mTORC1 在调节蛋白质合成、生长和增殖方面发挥着关键作用，以响应营养物质、生长因子和能量状态。mTORC1 的底物之一是 4E-BP1，mTORC1 对其磷酸化可逆转其对 eIF4E 的抑制作用，从而促进蛋白质合成。mTOR 复合物 1 中的 Raptor 被认为可以募集 4E-BP1，促进激酶 mTOR 对 4E-BP1 的磷酸化。我们应用化学交联结合质谱分析来深入了解 mTORC1 和 4E-BP1 之间的相互作用。使用交联试剂双[磺基琥珀酰亚胺]辛二酸酯，我们表明 Raptor 可以与 4E-BP1 交联。交联 Raptor-4E-BP1 的质谱分析导致鉴定出几个交联肽对。这些肽的编译表明，Raptor 的最 N 端 N 端保守结构域（特别是残基 89 到 180）是与 4E-BP1 相互作用的主要位点。在 4E-BP1 上，我们发现与 Raptor 的交联聚集在中央区域（氨基酸残基 56-72），我们称之为 RCR（Raptor 交联区域）。Raptor 的分子内交联表明 Raptor 存在两个结构区域：一个在 N 端区域，另一个在 C 端区域。支持 Raptor N 端保守结构域和 4E-BP1 中央区域紧密靠近的想法，我们发现包含 4E-BP1 的 RCR 的肽抑制 4E-BP1 与 Raptor 的交联和相互作用。此外，RCR 中的残基突变降低了 4E-BP1 作为 mTORC1 体外和体内底物的能力。
